Process for recovering cephalosporin C from aqueous solutions thereof

ABSTRACT

This invention relates to N-isobornyloxycarbonylcephalosporin C which is useful as an intermediate for the production of cephalosporin C and other cephalosporanic acid derivatives and is also useful as an antimicrobial agent.

United States Patent 1191 Horii et al. Dec. 2, 1975 I 1 PROCESS FORRECOVERING [58] Field of Search 260/243 C CEPHALOSPORIN C FROM AQUEOUSSOLUTIONS THEREOF [56] References Cited [75] Inventors: Satoshi Horii;Nariakira Mizokami, UNITED STATES PATENTS h f Osaka; Masahiko j3,160,631 12/1964 Peterson et a1. 260/243 c Hyogo; Susumu Shinagawa,Osaka; 3,167,550 l/l965 Chow et a1 260/243 C Michihiko Ochiai, Osaka;Tetsuya 3,454,564 7/1969 Vischer et a1, 260/243 c Okutani, Osaka, all ofJapan 3,573,296 3/1971 Johnson et a1. 260/243 C 3,738,978 6/1973 Jiigeret al. 260/112.5 Asslgneel Takeda h mi al In L 3,766,177 10/1973 Webberet a1. 260/243 0 Sa a FOREIGN PATENTS OR APPLICATIONS [22] Filed: 12,1973 2,016,703 10/1971 Germany 260/1 12.5 211 Appl. No.2 424,153

Primary ExaminerR. Gallagher I Related Apphcatlon Data AssistantExaminer-Diana G. Rivers Division Of SCI. NO. 229,169, 24, 1972, Pat.NO. Attorney, Agent or Firm Bu1'gc$$, Ryan and Wayne 3,819,619.

57 ABSTRACT [30] Foreign Application Priority Data 1 l b l h Feb. 26,1971 Japan 46-10063 f r.elat.es to "T oxycar. (my Cep alosporm C wh1ch1s useful as an 1ntermed1ate for the Feb. 26, 1971 Japan 46-10137producnon of cephalosporm C and other cephalospo- Aug. 17, 1971 Japan46-62417 a I ranlc acid der1vat1ves and 15 also useful as an ant1m1- 52US. Cl. 260/243 c; 260/463 cmbal agent [51 1 Int. Cl. C07D 501/12; CO7D501/26 7 Claims, N0 Drawings PROCESS FOR RECOVERING CEPHALOSPORIN C FROMAQUEOUS SOLUTIONS THEREOF CEPHEM COMPOUNDS The present invention relatesto cephem compounds. More specifically, the present invention relates toN- isobornyloxycarbonylcephalosporin C and to a method for theproduction thereof.

The present inventors have unexpectedly found thatN-isobomyloxycarbonylcephalosporin C is produced by reacting an aqueoussolution containing cephalosporin C with isobornyloxycarbonyl chloride.or an 1- isobornyloxycarbonylpyridinium chloride compound; that the thusproduced N-isobornyloxycarbonylcephalosporin C in the reaction mixtureis recoverable in high yield from the reaction mixture; and that theprotecting group i.e., the isobornyloxycarbonyl group, is easily removedby treating N-isobornyloxycarbonylcephalosporin C with an acid torecover cephalosporin C.

The present inventors have also found that the above producedN-isobornyloxycarbonylcephalosporin C is useful not only as anintermediate for the production of cephalosporin C and othercephalosporanic acid derivatives but also as an antimicrobial agent.

It has been found that when an aqueous solution containing cephalosporinC is reacted with an l-isobornyloxycarbonylpyridinium chloride compoundinstead of isobornyloxycarbonyl chloride, theN-isobornyloxycarbonylation of cephalosporin C proceeds under milderconditions and with improved efficiency.

Cephalosporin C can be recovered from a solution containingcephalosporin C, for example an aqueous solution obtained from orpresent in a culture broth of cephalosporin C producing microorganisms.

A known method for recovering cephalosporin C comprises the steps ofreacting cephalosporin C in an aqueous solution with sodium2,4,6-trinitrobenzenesulfonate and recovering the resulting2,4,6-trinitrophenylcephalosporin C. However, it is impossible to removethe group without incurring side reactions, e.g. the decomposition ofthe A3- cephem ring of the desired product.

It is an object of the present invention to provide a useful and novelcompound, i.e., N-isobornyloxycarbonylcephalosporin C.

It is another object of the present invention to provide an industriallyfeasible method for the production of said compound.

A further object of the present invention is to provide a highlyeffective method for recovering cephalosporin C from its aqueoussolution.

A further object of the present invention is to provide an advantageousmethod for producing 7-aminocephalosporanic acid from cephalosporin Cthrough the formation of N-isobornyloxycarbonylcephalosporin C andN-isobornyloxycarbonylcephalosporin C ester, deacylation thereof and anester clevage reaction.

N-isobornyloxycarbonylcephalosporin C is produced by reactingcephalosporin C with isobornyloxycarbonyl chloride or anl-isobornyloxycarbonylpyridinium chloride compound.

Referring to the latter compound, the pyridine ring ofl-isobornyloxycarbonylpyridinium chloride compound may be substituted byone or more of substituents which do not disturb the reaction andsuitable isobornyloxycarbonylpyridinium chloride compounds includel-isobornyloxycarbonyl-4-dimethylaminopyridinium chloride,l-isobornyloxycarbonyl-4-amino-pyridinium chloride, and the like.

It is advantageous that the substituents of the 1-isobornyloxycarbonylpyridinium compounds are located in the 2- or 4-position of the pyridine ring and these are electron-donating groupssuch as amino, an alkylated amino (e.g. methylamino, ethylamino,dimethylamino), hydroxy groups, and the like.

The l-isobornyloxycarbonylpyridinium chloride compound is superior tothe isobornyloxycarbonyl chloride in that whereas isobornyloxycarbonylchloride is sparingly soluble in water and reacts more readily in amixture of water and an organic solvent such as acetone, dioxane ortetrahydrofuran-than in an aqueous solution, the1-isobornyloxycarbonylpyridinium chloride compounds on the other handare water soluble and react very readily even in the absence of anorganic solvent in the reaction system.

The l-isobornyloxycarbonylpyridinium chloride compound is produced byreacting isobornyloxycarbonyl chloride with a corresponding pyridinecompound according to per se known means for the production ofquaternary pyridinium salts.

Each of the isobornyloxycarbonyl chloride and 1-isoborynlcarbonylpyridinium chloride compounds may be either in theracemic form or in an optically active form.

cephalosporin C may be employed in an aqueous solution thereof.Furthermore, the aqueous solution may be an aqueous solution obtainedfrom a culture broth of cephalosporin C-producing microorganisms (e.e.,a filtrate of the culture broth).

The culture broth can be prepared according to per se known methods.

The culture broth or its filtrate may contain, in large amounts,water-soluble impurities as well as fat-soluble impurities (e.e.cephalosporin N, cephalosporin P, antifoams, etc.). In this case, it isrecommended that the aqueous solution should be prepared by subjectingthe culture broth or its filtrate to conventional purificationprocedures such as chromatography, extraction by the use of an organicsolvent (e.e. ethyl acetate, methyl ethyl ketone or the like).

lsobornyloxycarbonyl chloride may be added to the reaction system in theform of a solution in a watermiscible organic solvent which does nottake part in the reaction. Such an organic solvent includes acetone,dioxane, tetrahydrofuran or the like.

The l-isobornyloxycarbonylpyridinium chloride compound may be suppliedto the reaction system in the form of an aqueous solution.

The reaction is suitably carried out in a suitable solvent. Such asolvent includes water, an organic solvent, particularly awater-miscible solvent (e.g. acetone, dioxane, tetrahydrofuran, etc.) ora mixture of such an organic solvent and water.

Usually it is advantageous to carry out the reaction at a pH value in arange from about 6.5 to 11; suitably from about 7 to 10, and mostsuitably from about 8 to 9.

As the pH adjusting agent, there may be employed an inorganic base, e.g.sodium hydrogen carbonate, so-

dium carbonate, sodium hydroxide, potassium hydroxide, lithiumhydroxide, barium hydroxide or magnesium oxide, or an organic base, e.g.triethylamine, tributylamine, N-methylmorpholine or pyridine.

The reaction may be conducted at temperatures varying from a lowtemperature to room temperature or, under certain circumstances, even atelevated temperatures. However, the reaction is suitably carried out ata temperature ranging from about C to 50C and more suitably from about0C to 35C.

Under the above reaction conditions, the reaction is usually completedwithin a period of time ranging from about minutes to about 4 hours.

The desired product, i.e., N-isobornyloxycarbonylcephalosporin C isrecovered from the reaction mixture, for example, by acidifying thereaction mixture with a mineral acid (e.g. hydrochloric acid, sulfuricacid, etc.) to a low pH (suitably a pH of about 2 to 3) and thensubjecting the resulting mixture to an extraction with an organicsolvent which is not freely miscible with water such as an ether (e.g.diethylether), an alcohol (e.g. n-butanol), an ester (e.g. ethylacetate), a ketone (e.g. methyl ethyl ketone) or a halogenatedhydrocarbon (e.g. methylene chloride). On evaporation of the solventfrom the extract, the desired product is obtained. When theconcentration of N-isobornyloxycarbonylcephalosporin C is relativelyhigh, N-isobornyloxycarbonylcephalosporin C may be recovered byprecipitation of the compound from the reaction mixture by adjusting theaqueous reaction mixture to an acid pH and suitably a pH of about 1 to 3with the mineral acid and collecting thus formed precipitates.

The thus obtained isobornyloxycarbonylcephalosporin C may further bepurified, by a procedure which is routinely used for the purification offat-soluble antibiotics and suitably by a procedure which is used forthe purification of acidic fat-soluble antibiotics, for example, by thesteps of extracting the active substance from the weakly acidifiedmixture with a suitable organic solvent, bringing 'the desired productinto an aqueous phase under alkaline conditions (suitably at a pH ofabout 8 to 9) by means of extraction and bringing it again into anorganic phase under acid conditions (suitably, pl-l of about 1 to 3).Aside from those procedures, one may, of course, employ such techniquesas countercurrent extraction, distribution, adsorption, ion exchange,molecular-sieve chromatography, etc. in suitable combinations to obtaina purified N-isobornyloxycarbonylcephalosporin C.

As mentioned above, a culture broth containing cephalosporin C may beemployed as a solution containing cephalosporin C. However, culturebroths containing cephalosporin C usually contain cephalosporin N aswell. To remove cephalosporin N from such a mixture, one may takeadvantage of the phenomenon that cephalosporin N is decomposed in acidsolutions according to per se known means as set out below.

For instance, before or after the reaction of such a solution withisobornyloxycarbonyl chloride or the lisobornyloxypyridinium chloridecompound, one may allow the solution to stand at a pH of about 3 and atabout 37C for about 2 hours to decompose the cephalosporin N. As analternative, one may take advantage of the fact that cephalosporin Nalone is decomposed at the next step when trifluoroacetic acid, forexample, is used to remove the isobornyloxycarbonyl group. Aside fromthose methods, one may of course separateisobornyloxycarbonylcephalosporin C fromisobornyloxycarbonylcephalosporin N by such conventional techniques aschromatography, counter-current extraction and the like.

Cephalosporin C is recovered by treating the thus produced and collectedN-isobornyloxycarbonylcephalosporin C with an acid.

The treatment with an acid is directed to the removal of the protectinggroup, i.e., isobornyloxycarbonyl, fromN-isobornyloxycarbonylcephalosporin C.

The reaction mixture may be at a low pH suitably at a pH less than about2.5, more suitably at a pH lower than about 2.0 and most suitably at apH less than about 1.0.

It is most advantageous that trifluoroacetic acid or a mixture oftrifluoroacetic acid and acetic acid is employed as the acid. Whentrifluoroacetic acid is employed, tha amount of the acid relative toN-isobornyloxycarbonylcephalosporin C is usually in a range from about10 to moles per mole of N-isobornyloxycarbonylcephalosporin C.

Use may be made of a reaction solvent which does not disturb thereaction that is, the solvent is substantially unreactive. Such asolvent is exemplified by substances as acetic acid, dichloromethane,dichloroethane, chloroform, dioxane, tetrahydrofuran, ether, ethylacetate, trifluoroacetic acid, and the like. It is preferable that thereaction is carried out in a mixture wherein the water content is lessthan about 30 percent (W/W), and the use of anhydrous or substantiallyanhydrous trifluoroacetic acid or trifluoroacetic acid havingadvantageously a water content of less than about 10 percent.

The time and temperature of the reaction and the amount of the acid areselected with a view to ensuring a complete cleavage under the mildestpossible conditions. For example, when anhydrous trifluoroacetic acid isemployed, the optimum conditions are about 10 to 25C and about 10 to 30minutes.

After the above reaction, the desired cephalosporin C is recovered fromthe reaction mixture by per se well known means such as concentration,countercurrent extraction, chromatography using a molecular-sieve or anion exchange resin, and the like.

Thus, according to the method of the present invention, cephalosporin Cin a crude solution can be easily recovered as pure cephalosporin C inhigh yield.

Those skilled in the art will readily understand modifications of thepresent method for the recovery of cephalosporin C, for example, byadding further per se conventional means to the above mentionedtechniques, and it should be noted that those modifications fall withinthe scope of the present invention.

It is to be noted that according to the present invention7-aminocephalosporanic acid may be produced fromN-isobomyloxycarbonylcephalosporin C by esterifyingN-isobornyloxycarbonylcephalosporin C to produceN-isobornyloxycarbonylcephalosporin C ester and subjecting the thusproduced ester to a deacylation reaction and to an ester clevagereaction.

For this purpose, N-isobornyloxycarbonylcephalosporin C is firstsubjected to an esterification reaction which is conventional per se.Preferably the ester is one which, after the deacylation reaction, canbe cleaved to give rise again to a free carboxyl group under mildconditions. For example, the dibenzhydryl ester which can be obtained bya reaction with diphenyldiazomethane can be easily cleaved with, forexample, trifluoroacetic acid and, as such, is useful in the practice ofthis reaction. Aside from the above ester, the ester obtainable byreacting with any one of the following agents can be easily cleaved byhydrolysis or alcoholysis and, as such, are of use in this reaction. Theesters referred to above are those obtainable with, for example,silylating agents, e.g. trialkylhalogenosilane,triaralkylhalogenosilane, trialkoxyhalogenosilane,trialkylhalogenosilane, trialkoxyhalogenosilane,tetralkyldiaryldisilazane, hexalkyldisilazane, hexaryldisilazane etc.;silenating agents, e.g. dialkyldihalogenosilane,dialkoxydihalogenosilane, alkyltrihalogenosilane,alkoxytrihalogenosilane, diaralkyldihalogenosilane,hexalkylcyclotrisilazane, octalkylcyclotetrasilazane,octaralkylcyclotetrasilazane, etc.; bis(trialkyltin) oxide,N-trialkylstannyldialkylamine, trialkylstannylalkoxide,tri-n-alkyl(alkylthio)tin, etc. The alkyl group may be eitherstraight-chain or branched, e.g. methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, t-butyL i-butyI, npentyl, n-hexyl, dodecyl, decyl,etc.; cycloalkyl is exemplified by cyclohexyl, cyclopentyl, etc.;aralkyl is exempliiied by benzyl, phenethyl, etc.; aryl is exemplifiedby phenyl, tolyl, p-chlorophenyl, etc.; alkoxy is exemplified bymethoxy, ethoxy, propoxy, etc.

Referring to the deacylation reaction,Nisobornyloxycarbonylcephalosporin C ester prepared in the above manneris subjected to a deacylation reaction. In the first place, theaforesaid ester is reacted with a halogenating agent (e.g. POCI PCIetc.) to obtain the corresponding iminohalide compound. This reaction isdesirably conducted in an anhydrous inert solvent, and it is alsopreferable to employ an acid acceptor which is able to neutralize theacid which is formed in the course of the reaction. For instance, it isdesirable to use 1.1 moles of phosphorous pentachloride and 5 moles ofpyridine per mole of the ester. This reaction may also be effected byadding the aforesaid halogenating agent to the esterification reactionsystem.

The reaction temperature is not critical, but to inhibit side reactions,this reaction is suitably carried out at a low temperature, for example,between about 40 and l0C.

The resulting iminohalide compound is then reacted with an alcohol toproduce the corresponding iminoether compound. The alcohol is ordinarilyan aliphatic or aryl alcohol of up to 14 carbon atoms, exemplaryalcohols including methanol, ethanol, propanol, amyl alcohol, butanol,benzyl alcohol, 2-phenylethanol, and the like. This reaction ispreferably conducted in the presence of an acid acceptor such as, forexample, a tertiary amine. For this purpose, one may add the requiredamount of acid acceptor in this reaction step or add or excess of theacceptor in the esterification or halogenation step. Like theimmediately preceding reaction step, this reaction is also desirablyconducted at a low temperature, for example at about to 0C. The alcoholmay be used in excess when it is also expected to cleave the silylgroup. The resulting iminoether compound is then subjected to ahydrolysis reaction to produce 7-aminocephalosporanic acid ester. Thereaction may be carried out under acidic or alkaline conditions. Thus,the reaction may be carried out in the presence of an inorganic acid(e.g. hydrochloric acid, sulfuric acid, phosphoric acid or the like), anorganic acid (e.g. p-toluenesulfonic acid, trifluoroacetic acid, formicacid, or the like), a basic material (e.g. sodium bicarbonate, etc.)according to a per se known means as described for example in BritishPat.

Thus obtained 7-aminocephalosporanic acid ester is subjected to a per seknown suitable ester cleavage reaction by the procedure described forexample in British Pat. No. 1,041,985. The reaction is usually carriedout by treating 7-aminocephalosporanic acid ester with trifluoroaceticacid, if desired, in an organic solvent (e.g. anisole, etc.), or byreducing '7-aminocephalosporanic acid ester according to a conventionalmeans, for example, by use of palladium, platinum or a like catalyst.However it is to be noted that when the ester group of the abovementioned iminohalide compound is a silyl ester group or a silenic estergroup, such as ester group may be removed by use of a large amount ofthe alcohol in the above reaction step for the formation of saidiminoether compound, or alternatively such an ester group may be removedin the hydrolysis reaction step to effect the deacylation reaction. Thevariations of such procedures also obviously falls within the scope ofthe present invention.

After the reaction, the desired 7-aminocephalosporanic acid is recoveredby adjusting the pH of the reaction mixture to or substantially near theisoelectric point of 7-aminocephalosporanic acid.

The products obtained in the foregoing manner, i.e.,N-isobornyloxycarbonylcephalosporin C and cephalosporin C, both haveantimicrobial properties and are not only therapeutically useful but arealso useful as intermediates in the manufacture of syntheticcephalosporins such as 7-aminocephalosporanic acid and other compounds.It need not be mentioned that 7- aminocephalosporanic acid, which can bederived from Nisobornyloxycarbonylcephalosporin C, is useful as astarting material for the production of synthetic cephalosporins. Thoughthe above description has been made with particular reference to theacid forms, those compounds are capable of forming salts with suchmetals as sodium, potassium, calcium, aluminum, and the like, and withsuch organic bases as trimethylamine, triethylamine, tributylamine,triamylamine, pyridine and the like.

This invention will be further illustrated by way of Examples whereinthe term part(s) by weight bears the same relationship to the termpart(s) by volume as do gram(s) to milliliter(s).

The term milliliter(s) may be abbreviated as ml..

REFERENCE EXAMPLE 1 Preparation of isobornyloxycarbonyl chloride:

In 200 parts by volume of dry ether is dissolved 61.6 parts by weight ofisoborneol and, under cooling with ice, a solution of 60 parts by weightof phosgene in parts by volume of tetrahydrofuran is added dropwise.After the dropwise addition has been completed, the mixture is stirredat 50C for 2 hours. The solvent is distilled off and the viscous oilyresidue is subjected to thin-layer chromatography on silica gel usingbenzene as a solvent. The resulting chromatogram attests to a completedisappearance of the starting material isoborneol and the formation of anew product having a higher Rf value. The IR absorption spectrum of thisproduct shows that it is the desired chloroforrnate. (Yield 83.2 partsby weight (95.6 percent)).

REFERENCE EXAMPLE 2 In 50 parts by volume of dry methylene chloride isdissolved 2.44 parts by weight of 4-dimethylamiopyridine and,accompanied by stirring, a solution of 4.65

7 parts by weight of isobornyloxycarbonyl chloride in dry methylenechloride is added.

After stirring at room temperature for 30 minutes, dry ether is added tothe mixture and the precipitate thus formed thereupon is then recoveredby filtration. After washing with dry ether, the precipitate isreprecipitated from dry methylene chloride-dry ether, whereupon 6.1parts by weight (yield 90 percent) of1-isobornyloxycarbonyl-4-dimethylaminopyridinium chloride is obtained.m.p. 7677C (decomp.); IR spectrum: max (KBr), 1780 cm, 1645 cm; UVspectrum: A max (H 297 mu 20400).

EXAMPLE 1 Preparation of N-isobornyloxycarbonylcephalosporin C Undercooling with ice, 0.4 part by weight of cephalosporin C sodium salt isdissolved in 0.87 parts by volume of a 1N aqueous solution of sodiumhydroxide, followed by the addition of 5 parts by volume of water and 2parts by volume of tetrahydrofuran. Then, 0.204 part by weight ofisobornyloxycarbonyl chloride and a 1N aqueous solution of sodiumhydroxide are added and, while the mixture is maintained under weaklyalkaline conditions, i.e., a pH of about 8, the mixture is stirred atroom temperature for 2.5 hours. After the reaction has been completed,the mixture is washed three times with 30 parts by volume of ethylacetate. Under cooling with ice, the aqueous layer is adjusted to pH 3with 1N hydrochloric acid and extracted three times with 50 parts byvolume of ethyl acetate. The ethyl acetate layer is washed with asaturated aqueous solution of sodium chloride and dried over anhydroussodium sulfate. The solvent is distilled off and the residue isreprecipitated from ethyl acetate-petroleum ether. The procedure yields0.460 part by weight (88.6 percent) of the desired product as awhitepowder. m.p. 118120C. Thin-layer chromatography (on silica gel);ethyl acetate: methanol (2.3), Rf 0.42. Ultraviolet absorption spectrum(methanol): 262 p. (e 7500). Infrared absorption spectrum (KBr)cm 1790,1720 Elemental analysis: for C H O N S: C, 54.44 H, 6.26: N, 7.06(percent); Found: C, 54.45: H, 6.42: N, 6.64 (percent).

EXAMPLE 2 A culture filtrate containing 1,500 /ml. of cephalosporin C(20,000 parts by volume) is extracted with ethyl acetate (3 times with Avolume) to remove the fat-soluble impurities and the aqueous layer isadjusted to pH 8.5 with an aqueous solution of sodium hydroxide.Accompanied by stirring 30 parts by weight of isobornyloxycarbonylchloride is added and while the reaction mixture is maintained at about8.5 with a solution of sodium hydroxide, the reaction is allowed toproceed at room temperature for about 2 hours. After the reaction, thereaction mixture is made acidic (pH 2) with hydrochloric acid andextracted three times with 5000 parts by volume of ethyl acetate. Theresulting ethyl acetate extract is further extracted with 0.2M phosphatebuffer (pH 8). The aqueous layer is adjusted to pH 3 and theisobornyloxycarbonylcephalosporin C is brought again into ethyl acetate.

The ethyl acetate solution thus obtained is washed with water, dried andconcentrated under reduced pressure. Finally, petroleum ether is addedto the concentrate, whereupon 56.6 parts by weight of crudeisobornyloxycarbonylcephalosporin C is obtained.

EXAMPLE 3 In 50 parts of volume of water is dissolved 9.4 parts byweight of a crude preparation containing 45% (W/V) of cephalosporin Cand, under cooling with ice, the solution is adjusted to pH 8-9 with 1Nsodium hydroxide. To this solution is added a solution of 4.0 parts byweight of isobornyloxycarbonyl chloride in tetrahydrofuran. Whilestirring, the mixture is allowed to react at room temperature for 2hours, the pH of the reaction mixture being maintained at pH 8 to 9 with1N sodium hydroxide. The reaction mixture is washed with ethyl ether andthe aqueous layer is made acidic with IN bydrochloric acid. Theresulting precipitate is extracted with ethyl acetate and, in the mannerdescribed in Example 2, an extraction is carried out with a 0.2Mphosphate buffer. The final ethyl acetate extract is concentrated,followed by the addition of petroleum ether. The procedure yields 4.8parts by weight of crude isobornyloxycarbonylcephalosporin C.

The thin layer chromatogram (TLC) of the above product (silica gel;solvent system: ethyl acetate: methanol 2:3) reveals a spot ofisobornyloxycarbonylcephalosporin C at Rf 0.42 and a faint spot at Rf0.18.

To obtain a pure preparation of isobornyloxycarbonylcephalosporin C, theabove product is further purified by chromatography on silica gel(solvent system: methanol: ethyl acetate in a ratio of 3:2) to obtainthe same product as in Example 1. Antibacterial activity of the product(minimal inhibitory concentration; assay organism: Bacillus subtilis)12.5 y/ml.

EXAMPLE 4 In 50 parts by volume of anisole is dissolved 10 parts byweight of the N-isobornyloxycarbonylcephalosporin C obtained in Example3 and, under substantially anhydrous conditions and at 20C, parts byvolume of trifluoroacetic acid is added. The mixture is further stirredat 20C for 5 minutes and at room temperature for 20 minutes. Then, themixture is neutralized with triethylamine to pH 6, followed by theaddition of water. The resulting aqueous solution is purified bychromatography on a strongly basic anion exchange resin, e.g. Dowex l(manufactured and sold by the Dow Chemical Co., Midland, Michigan, USA.)by washing with water, eluting with 0.2M ammonium acetate buffer, pH 5.8and, then, to chromatography on activated carbon. Fractions containingcephalosporin C are collected and adjusted to pH 6.5 with N-NaOH,followed by the addition of methanol to yield crystals. The procedureyields 4.7 parts by weight of crystals of cephalosporin C sodium salt.

EXAMPLE 5 In 2 parts by volume of anisole is dissolved 1.1 part byweight of N-isobomyloxycarbonylcephalosporin C and, at 20C, 7 parts byvolume of trifluoroacetic acid is added. Under substantially anhydrousconditions, the mixture is stirred at the aforesaid temperature for 5minutes and at room temperature for 30 minutes. Anhydrous ethyl ether isthen added. The resulting precipitate is recovered by filtration, washedwell with dry ethyl ether and allowed to stand overnight, under reducedpressure, in a desiccator containing potassium hydroxide and phosphoricanhydride. The resulting powder is dissolved in water and the solutionis adjusted to pH 6.5 with 1N sodium hydroxide, followed by the additionof ethanol. The procedure yields 0.5

part by weight of crystals of cephalosporin C sodium salt (60 percent).

EXAMPLE 6 In 10 parts by volume of methanol is dissolved 1.0 part byweight of N-isobornyloxycarbonylcephalosporin C and, under cooling withice, 1.1 part by weight of diphenyldiazomethane is added. The mixture isstirred under cooling with ice for 30 minutes and, then, at roomtemperature for 3 hours, after which time the methanol is distilled offunder reduced pressure.

The residue is dissolved in 150 parts by volume of ethyl acetate and theethyl acetate layer is washed with 0.5 N sodium hydrogen carbonate andwith water. The ethyl acetate layer is dehydrated, and the solvent isdistilled off. The residue is purified by chromatography on silica gel(solvent system; ether: chloroform 1:1), whereupon 1.5 part by weight(90 percent) of isobornyloxycarbonylcephalosporin C dibenzyhydryl esteris obtained. m.p. 98-99C; elemental analysis: calculated for C l-1 19 8:C, 68.59; H, 6.19; N, 4.53; S, 3.45; found: C, 68.97; H, 6.37; N, 4.23;S, 3.40. Ultraviolet absorption spectrum (in methanol): )t max 260 p.(c=8,l00). Infrared absorption spectrum (KBr): characteristicabsorptions at 1790, 1750 and 1730 cm". Thin-layer chromatography(silica gel; ethyl acetatechloroform 1:4): Rf 0.42.

In 30 parts by volume of anhydrous methylene chloride is dissolved 1.0part by weight of the dibenzhydryl ester obtained above and, whilecooling at -l to 20C, 0.5 part by volume of anhydrous pyridine and partsby volume of a solution of 0.7 part by weight phosphorus pentachloridein anhydrous methylene chloride are added dropwise in the ordermentioned. While cooling, the mixture is reacted for 1 hour. After theaddition of 7 parts by volume of anhydrous methanol, the mixture isstirred at -l5 to 20C for 40 minutes and at room temperature for 1 hour,after which time 11 parts by volume of 2N hydrochloric acid is added,and stirred for 45 minutes. After adjusting the pH of the mixture to 7.9with lN-NaOH, the methylene chloride layer is separated. The aqueouslayer is further extracted with methylene chloride. The two extracts arecombined and the solvent is distilled off under reduced pressure. Theresidue is dissolved in 5 parts by volume of anisole and, in the absenceof moisture and under cooling (20C), 14 parts by volume oftrifluoroacetic acid is added. The reaction is conducted, while cooling,for 5 minutes and at room temperature for 20 minutes. After the reactionmixture is neutralized (pH 6) with triethylamine, water is added. Themixture is passed through a column of a strong anion exchange resin,e.g. Dowes l X 2 (in the form of the acetate which is manufactured andsold by the Dow Chemical Co., Midland, Michigan, U.S.A.). After thecolumn is washed with water, the adsorbed 7-amino-cephalosporanic acidis eluted with 0.5 N acetic acid. The eluate is lyophilized. There isobtained a yield of 0.205 part by weight (70 percent).

EXAMPLE 7 In 30 parts by volume of methylene chloride is suspended 2.79parts by weight of the crude N-isobornyloxycarbonylcephalosporin Cobtained in Example 3, followed by the addition of 1.8 part by volume ofpyridine. While cooling with ice, 0.67 part by weight ofdimethoxydichlorosilane is added. The mixture is stirred for 30 minutes.The reaction mixture which has 10 now become clear is cooled to 40C anda solution of 1.71 part by weight of phorphorus pentachloride in 25parts by weight of methylene chloride is added dropwise over an 8 minuteperiod. Then, the temperature is gradually elevated and, at -15 to 20C,the mixture is stirred for 2 hours. The thin layer chromatogram of thereaction product attests to the disappearance of the starting materialand the emergence of a spot corresponding to a new chlorination product.The reaction mixture is chilled again to 40C and 10 parts by volume ofmethyl alcohol is added. The temperature is gradually elevated and, at20 to 15C, the mixture is stirred for 2.5 hours, followed by theaddition of 1.2 parts by volume of pyridine and, then, 6 parts by volumeof pure water. The mixture is stirred at 20 to 0C for 15 minutes and at0 to 5C for 15 minutes. The insoluble materials which separate from thereaction mixture are filtered off and the aqueous layer is separated,washed with ether and adjusted to pH 3.5 with a saturated aqueoussolution of sodium hydrogen carbonate, whereupon colorless crystals of7-aminocephalosporanic acid are obtained. The crystals are recovered byfiltration, washed twice with cold water and dried.-

Using this procedure, 7-aminocephalosporanic acid is obtained in highyield. As determined by melting point measurement, thin layerchromatography and infrared spectrophotometry, this product is identicalwith an authentic sample of 7-aminocephalosporanic acid.

EXAMPLE 8 In 14 parts by volume of methylene chloride is suspended 1.37parts by weight of the crude N-isobornyloxycarbonylcephalosporin Cprepared in Example 3 and, at room temperature, 9.54 parts by weight oftriethylamine and 0.46 part by weight of dimethyldichlorosilane areadded. The mixture is stirred for 30 minutes. To the clear solution thusobtained is added 0.6 part by weight of pyridine, and at 30C, a solutionof 0.57 part by weight of phosphorus pentachloride in 10 parts by volumeof methylene chloride is added dropwise over 10 minutes. The mixture isstirred at 30 to l 5C for 2 hours. Then, the mixture is cooled again to30C and 5 parts by volume of methanol is added. The mixture is stirredat 20 to -l5C for 2 hours. At 20C, 0.4 part by volume of pyridine and 4parts by volume of pure water are added. Then, the temperature isgradually elevated and, at 5C, the mixture is stirred for 40 minutes.The aqueous layer is separated and adjusted to a pH of 3.5 with asaturated aqueous solution of sodium hydrogen carbonate, whereuponcrystals separate out. The crystals are recovered by filtration, washedwith cold water and dried. In the above manner, 7-aminocephalosporanicacid is obtained in good yield. Based on its infrared and otheridentification data, this product is identical with an authentic sampleof 7-aminocephalosporanic acid.

EXAMPLE 9 A culture broth containing cephalosporin C is adjusted to a pHof 5.5 and the mycelia are removed by a pH of 8.5 with a solution ofsodium hydroxide. To this solution is added 50 parts by weight ofl-isobornyloxycarbonyl-4dimethylamino-pyridinium chloride and, at roomtemperature, the reaction is allowed to proceed for about 1.5 hours, thepH of the solution being controlled at a pH of 8.0 to 8.5.

After the reaction is completed, the mixture is made acidic (pH 2) withhydrochloric acid and, then, extracted three times with 500 parts byvolume portions of ethyl acetate. The resulting ethyl acetate solutionis extracted with 0.2M phosphate buffer (pH 8). The aqueous layer isadjusted again to pH 2 and the N- isobornyloxycarbonylcephalosporin C isbrought into ethyl acetate. The ethyl acetate solution thus obtained iswashed with a saturated aqueous solution of sodium chloride and, then,dried over anhydrous sodium sulfate. The solvent is distilled off underreduced pressure and petroleum ether is added to the residue, whereupon1 1.4 parts by weight of crude N-isobornyloxycarbonylcephalosporin C isobtained.

The purity of this crude product is about 66 percent as measured by abioassay method with Bacillus subtilis as an assay organism.

The above crude product is chromatographed on acid-treated silica gelusing chloroform and ethyl acetate (1:1) as a solvent system, whereupon3.4 parts by weight of pure N-isobornyloxycarbonylcephalosporin C isobtained. The biological and physicochemical properties of this productare in complete agreement with those of theNisobornyloxycarbonylcephalosporin C synthesized by the procedure ofExample 3.

EXAMPLE 10 In 25 parts by volume of water is dissolved 4.73 parts byweight of crystalline cephalosporin C sodium salt, followed by theaddition of 10 parts by volume 1N- sodium hydrogen carbonate and, then,4.0 parts by weight of 1isobornyloxycarbonyl-4-dimethylaminopyridiniumchloride. Accompanied by stirring, the mixture is allowed to react atroom temperature for 1.5 hour.

After the reaction is completed, the mixture is adjusted to a pH of 2with 2N hydrochloric acid and, then, extracted with ethyl acetate. Theethyl acetate layer is washed with water, dried over anhydrous sodiumsulfate and concentrated under reduced pressure.

To the concentrate is added petroleum ether, whereupon 5.1 parts byweight of the desired compound N- isobornyloxycarbonylcephalosporin C isobtained (yield 85.6 percent). Thin layer chromatography, ultravioletabsorption spectrum, and infrared absorption spectrum show the productto be identical with the product of Example 1. Antibacterial potency(minimal inhibitory concentration) of the product: 12.5 'y/ml againstBacillus subtilis.

EXAMPLE 1 l A culture broth (2,000 parts by volume) containingcephalosporin C is passed through a column of an anion exchange resine.g. Amberlite lRC-50 (H-form) manufactured and sold by Rohm and HaasU.S.A., and 35 parts by weight of 1-isobornyloxycarbonyl-4-aminopyridinium chloride is added to the effluent. The mixture isstirred at room temperature for 1.5 hour, the pH of the mixture beingmaintained within the range of 8.0 to 8.5. The resulting reactionmixture is brought to a pH 2 with 2N hydrochloric acid and, then,extracted with ethyl acetate. The ethyl acetate layer is washed withwater and extracted with 0.2M phosphate buffer (pH 8.0). The aqueouslayer is adjusted again to a pH of 2 and extracted with ethyl acetate.The ethyl acetate layer is washed with a saturated aqueous solution ofsodium chloride and dried over anhydrous sodium sulfate, followed by theremoval of ethyl acetate by distillation. Petroleum ether is then addedto the residue, whereupon 6.9 parts by weight of crudeN-isobornyloxycarbonylcephalosporin C is obtained.

As measured by a bioassay method using Bacillus subtilis as an assayorganism, the purity of this product is about 54 percent.

EXAMPLE 12 10.2 Parts by weight of N-isobornyloxycarbonylcephalosporin Cis added to a mixture of 700 parts by volume dried methylene chlorideand 6.4 parts by volume of dried pyridine, followed by the addition of12.3 parts by volume of trimethylchlorosilane. The resulting mixture isstirred at room temperature for 1 hour. After the reaction, the reactionmixture is cooled at -20C, and to the reaction mixture is added 21.7parts by volume of pyridine and 14.2 parts by weight of phosphoruspentachloride dissolved in 500 parts by volume of dried methylenechloride. The resulting mixture is stirred at 12C for 45 minutes, andafter the addition of 250 parts by volume of dry methanol at 10C, thestirring is further continued at 10C for 30 minutes and at 25C for 30minutes. To the reaction mixture is added 40 parts by volume of a 25percent (W/V) formic acid, and the resulting mixture is adjusted to a pHof 2.0 by the addition of triethylamine. The resulting mixture isstirred for 45 minutes. The pH of the reaction mixture is then adjustedto 3.5 by the addition of triethylamine, and the resulting mixture iskept standing at 0C resulting in the formation of precipitates. Theprecipitates are collected by filtration, washed with methylenechloride, methanol, and ether in this order, and dried to obtain 3.6parts by weight of 7-aminocephalosporanic acid. The yield is 77.3percent.

What is claimed is:

1. A process for recovering cephalosporin C from an aqueous solutionthereof, which comprises reacting said solution withisobornyloxycarbonyl chloride or an isobornyloxycarbonylpyridiniumchloride compound to produce the correspondingN-isobornyloxycarbonylcephalosporin C, recovering saidN-isobornyloxycarbonylcephalosporin C by extraction or precipitation,and removing the protective isobornyloxycarbonyl group by adding an acidto give a pH of less than about 2.5 to yield said cephalosporin C.

2. A process according to claim 1, wherein the solution containingcephalosporin C is an aqueous solution obtained from a culture broth ofcephalosporin C-producing micro-organisms.

3. A process according to claim 1, the treatment of theN-isobomyloxycarbonylcephalosporin C is effected by the addition oftrifluoroacetic acid or a mixture of trifluoroacetic acid and aceticacid.

4. A process according to claim 3, wherein the water content of thetrifluoroacetic acid does not exceed about 10 percent.

5. A process according to claim 1, wherein the step for the removal ofthe protective group of N-isobornyloxycarbonylcephalosporin C by addingan acid is conducted under substantially anhydrous conditions.

6. A process according to claim 3, wherein the amount of trifluoroaceticacid added is about 1 part by weight to about 40 parts by weight oftrifluoroacetic ny]oxycarbonylcephalosporin C is treated withtrifluoacid per 1 part by weight of N-isobornyloxycarbonylroacetic acidat a temperature ranging from about cephalosporin C. to 25C.

7. A process according to claim 3, wherein N-isobor-

1. A PROCESS FOR RECOVERING CEPHALOSPORIN C FROM AN AQUEOUS SOLUTIONTHEREOF, WHICH COMPRISES REACTING SAID SOLUTION WITHISOBORNYLOXYCARBONYL CHLORIDE OR AN ISOBORNYLOXYCARBONYLPYRIDINIUMCHLORIDE COMPOUND TO PRODUCE THE CORRESPONDINGN-ISOBORNYLOXYCARBONYLCEPHALOSPORIN C, RECOVERING SAIDN-ISOBORNYLOXYCARBONYLCEPHALOSORIN C BY EXTRACTION OR PRECIPITATION, ANDREMOVING THE PROTECTIVE ISOBORNYLOXYCARBONYL GROUP BY ADDING AN ACID TOGIVE A PH OF LESS THAN ABOUT 2.5 TO YIELD SAID CEPHALOSPORIN C.
 2. Aprocess according to claim 1, wherein the solution containingcephalosporin C is an aqueous solution obTained from a culture broth ofcephalosporin C-producing micro-organisms.
 3. A process according toclaim 1, the treatment of the N-isobornyloxycarbonylcephalosporin C iseffected by the addition of trifluoroacetic acid or a mixture oftrifluoroacetic acid and acetic acid.
 4. A process according to claim 3,wherein the water content of the trifluoroacetic acid does not exceedabout 10 percent.
 5. A process according to claim 1, wherein the stepfor the removal of the protective group ofN-isobornyloxycarbonylcephalosporin C by adding an acid is conductedunder substantially anhydrous conditions.
 6. A process according toclaim 3, wherein the amount of trifluoroacetic acid added is about 1part by weight to about 40 parts by weight of trifluoroacetic acid per 1part by weight of N-isobornyloxycarbonylcephalosporin C.
 7. A processaccording to claim 3, wherein N-isobornyloxycarbonylcephalosporin C istreated with trifluoroacetic acid at a temperature ranging from about10* to 25*C.